Project Summary/Abstract: Arsenic is the most pervasive toxin, considered by the EPA to be one the most significant potential environmental threats to human health. Arsenic exposure is a cause of cancer, heart disease, childhood developmental delay, and disrupts the human microbiome. Our research program blossomed during the current funding period of our NIGMS grant, focusing on arsenic transporters and biotransformations, which modify its availability, speciation, mobility and toxicity. We are uniquely qualified for this project: over the lifetime of this grant, my group identified and characterized the majority of ars genes/proteins involved in arsenic transport, biotransformations and resistance and their impact on the global arsenic biogeocycle. We discovered enzymes of the arsenic methylation cycle and elucidated mechanisms and structures of the enzymes of biotransformation, developed biosensors for organoarsenicals herbicides and discovered organoarsenicals with the potential to be novel antimicrobial agents. My goals for the next five years fall into four categories. 1) Structure/function analysis of enzymes of arsenic biotransformations. We will elucidate the catalytic cycle of the ArsM arsenite S- adenosylmethione (SAM) methyltransferase, the ArsH methylarsenite oxidases, the ArsI C-As bond lyases and the ArsN N-acetyltransferase through biochemical and structural analysis. 2) Regulation and biosensing. We will determine the structural details of metalloregulation. We will devise new applications for sensing environmental organoarsenical pollutants. 3) Arsenic transporters; we identified a number of new permeases for organoarsenicals and will determine the mechanism of transport by a combination of molecular genetics, biochemistry and crystallography. 4) Arsenical antibiotics; we recently identified two organoarsenical natural products with antibiotic activity. We will determine the pathways of synthesis and mode of action of these novel compounds and discover new natural products with potential health applications. My overall vision is a research program of sufficient breadth to encompass identification of the physiological roles of known arsenic resistance genes and sufficient depth to elucidate their molecular mechanisms. Microbial genomes have many uncharacterized arsenic-related genes. There are predicted permeases and enzymes with no known substrate or function. We predict these are involved in arsenical transport or biotransformations. We will mine microbial genomes for new ars genes, deduce their evolutionary histories and determine how they affect cycling of environmental arsenicals. We will discover their physiological functions. Their protein products will be purified and characterized by biochemical and structural analyses. My overarching theme is to make substantial contributions to understanding of the global arsenic biogeocycle and its impact on human health.